High affinity fusion proteins and therapeutic and diagnostic methods for use

ABSTRACT

High affinity fusion proteins capable of binding and inhibiting the activity of soluble, interacting proteins (“SIPs”) are described. In specific embodiments the fusion proteins are multimers, preferably dimers, of SIP-specific fusion polypeptides which comprise SIP binding domains derived from SIP targets and/or anti-SIP immunoglobulin domains, as well as multimerizing components.

BACKGROUND

1. Field of the Invention

This invention relates to fusion proteins and multimeric fusion proteins (designated herein as “trapbodies”) with increased affinity for soluble, interacting proteins (“SIPs”), methods of producing such fusion proteins, and methods for treating, diagnosing, or monitoring diseases or conditions in which regulation of SIP molecules is desired.

2. Description of Related Art

Multimeric proteins that efficiently bind cytokines are described in U.S. Pat. No. 6,472,179. The use of cytokine receptor components in fusion proteins that bind cytokines has also been described in WO 96/11213 and WO 93/10151.

BRIEF SUMMARY OF THE INVENTION

The present invention provides fusion proteins capable of binding soluble, interacting proteins (“SIPs”) and preventing or inhibiting the SIPs from interacting with SIP targets. The fusion proteins, which, in specific embodiments are multimers designated herein as “trapbodies,” are useful for reducing, preventing, ameliorating or inhibiting conditions or diseases caused by normal or elevated levels of SIP molecules. The fusion proteins and trapbodies of the invention are further useful in a variety of in vitro and in vivo diagnostic and prognostic assays.

Accordingly, in a first aspect, the invention features a SIP-specific fusion polypeptide comprising (i) one or more components which comprise a SIP binding domain of a SIP target (“target binding domain or TBD”); (ii) one or more components which comprise a SIP binding domain of an immunoglobulin (“immunoglobulin binding domain or IBD”) and (iii) a multimerizing (M) component (TBD)_(x)-(IBD)_(y)-M, wherein x≧1 and y≧1. The multimerizing component is capable of interacting with another multimerizing component to form a higher order structure, e.g., a dimer, a trimer, etc. The multimer of the SIP-specific fusion polypeptides is termed a “trapbody” and is capable of binding to a SIP. The isolated nucleic acid molecule of the invention may further optionally comprise a signal sequence (SS) component. The signal sequence may comprise any sequence known to a skilled artisan for directing secretion of a polypeptide or protein from a cell, including recombinant, synthetic or natural sequences from any source, for example, from a secreted or membrane bound protein. Generally, a signal sequence is placed at the beginning or amino-terminus of the fusion polypeptide of the invention. In a preferred embodiment, the fusion polypeptide of the invention comprises one TBD component, one IBD component, and M (TBD-IBD-M).

In one embodiment, the SIP is a cytokine, and the SIP target is a cytokine receptor. In one aspect of this embodiment, the SIP target binding domain(s) is (are) derived from the specificity-determining component of a cytokine receptor protein or a cytokine binding fragment or derivative thereof. As used herein, a specificity-determining component of a receptor is one that specifically binds a cytokine, leading to its interaction with a second receptor component which is a signaling receptor component. The specificity-determining components of cytokine receptors that are useful for practicing this invention, include, but are not limited to, those set forth in U.S. Pat. No. 6,472,179. These include the specificity-determining components of receptors for interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-13, IL-15, IL-18, granulocyte macrophage colony stimulating factor, oncostatin M, leukemia inhibitory factor, ciliary neurotrophic factor, interferon-gamma, and cardiotrophin-1. In an embodiment wherein the SIP is IL-1, the specificity-determining component is a specificity-determining component of the IL-1 receptor or a fragment or derivative thereof. In a more specific embodiment, the IL-1 receptor protein is the cytokine binding portion of the IL-1R1 Type I or Type II protein. In another embodiment, the target molecule is IL-4, and the cytokine receptor protein is human IL-4Ra, or a fragment or derivative thereof.

In a related embodiment, the SIP target-binding domain is derived from a signaling component of a cytokine receptor or a cytokine binding fragment or derivative thereof. As used herein, a signaling component is defined as a receptor component that initiates cell signaling upon the interaction with another cytokine receptor component, which can be either a specificity-determining component or another signaling component of the cytokine receptor. A cytokine binding fragment or derivative of a signaling component is one that binds the cytokine alone or in the presence of a specificity-determining component of the receptor system. The signaling components of cytokine receptors that are useful for practicing this invention, include, but are not limited to, those set forth in U.S. Pat. No. 6,472,179. These include the signaling components of receptors for interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-13, IL-15, IL-18, granulocyte macrophage colony stimulating factor, oncostatin M, leukemia inhibitory factor, ciliary neurotrophic factor, MIF, interferon gamma and cardiotrophin-1. In one embodiment wherein the SIP is IL-1, the signaling component is a signaling component of the IL-1 receptor system, such as IL-1R accessory protein. In yet another embodiment, wherein the SIP is IL-4, the signaling component is IL-2Rgamma.

In a second aspect, the invention provides a SIP-specific fusion polypeptide comprising (i) two or more components which comprise a SIP binding domain of an immunoglobulin and (ii) a multimerizing (M) component. In specific embodiments, M is a multimerizing component multimerizes with a multimerizing component on another SIP-specific fusion polypeptide to form a trapbody capable of binding to a SIP. In this aspect of the invention, the two or more SIP binding domains of an immunoglobulin are immuno-specific for the same or different epitopes of the same SIP.

In a specific embodiment, when the SIP is human IL-6, the fusion polypeptide comprises IBD-IBD-M, each IBD comprising one or more sequences selected from the group consisting of sequences SEQ ID NOs:17-112.

In one embodiment, the components of the SIP-specific fusion polypeptides of the invention are connected directly to each other. In other embodiments, a spacer sequence may be included between one or more components, which may comprise one or more molecules, such as amino acids. For example, a spacer sequence may include one or more amino acids naturally connected to a fusion polypeptide component. A spacer sequence may also include a sequence used to enhance expression of the fusion protein, provide restriction sites, allow component domains to form optimal tertiary or quaternary structures and/or to enhance the interaction of a component with its SIP. In one embodiment, the SIP-specific fusion polypeptide of the invention comprises one or more peptide sequences between one or more components which is (are) between 1 and 100 amino acids. In a preferred embodiment, the peptide sequence is between 1 and 25 amino acids.

Further embodiments may include a signal sequence at the beginning or amino-terminus of a SIP-specific fusion polypeptide of the invention. Such a signal sequence may be native to the cell, recombinant, or synthetic.

The components of the SIP-specific fusion polypeptide of the invention may be arranged in a variety of configurations. For example, in certain embodiments, described from the beginning or amino-terminus of the fusion protein, one or more components comprising SIP target binding domain(s) (TBD) may be followed by one or more components comprising SIP-directed immunoglobulin binding domains (IBD), followed by a multimerizing component (M). Such a fusion protein may also optionally include a signal sequence (SS) prior to the one or more TBD or IBD(s).

Further configurations contemplated by the invention may be depicted as follows: (TBD)_(x)-(IBD)_(y)-M; (TBD)_(x)-M-(IBD)_(y); (IBD)_(x)-(TBD)_(y)-M; SS-(TBD)_(x)-(IBD)_(y)-M; SS-(IBD)_(x)-(TBD)_(y)-M; (IBD)_(x)-M-(IBD)_(y); SS-(IBD)_(x)-M-(IBD)_(y); (IBD)_(x)-(IBD)_(y)-M; SS-(IBD)_(x)-(IBD)_(y)-M; SS-M-(IBD)_(x)-(TBD)_(y); SS-M-(TBD)_(y)-(IBD)_(x); SS-M-(IBD)_(x)-(IBD)_(y); wherein x≧1 and y≧1. In each embodiment, the one or more IBDs or TBDs may be directed to the same or different epitopes of the receptor. In one specific embodiment, x=1-10 and y=1-10. In an even more specific embodiment, x=land y=1, or x=2 and y=2. In a preferred embodiment, the fusion polypeptide has the arrangement (IBD)_(x)-(IBD)_(y)-M, wherein the first and second IBD domains are immunospecific for different epitopes of the SIP, x=1 and y=1. In another preferred embodiment, the fusion polypeptide has the arrangement (IBD)_(x)-(TBD)_(y)-M wherein x=1 and y=1.

In a third aspect, the invention features a trapbody comprising a dimer of two SIP-specific fusion polypeptides of the invention formed by the interaction of the multimerizing components.

In a fifth aspect, the invention features a nucleic acid sequence encoding a SIP-specific fusion polypeptide which comprises (i) one or more components which comprise a SIP binding domain of a SIP target; (ii) one or more components which comprise a SIP binding domain of an immunoglobulin and (iii) a multimerizing (M) component. In specific embodiments, the M is a multimerizing component that multimerizes with a multimerizing component on another SIP-specific fusion polypeptide to form a multimer of the fusion polypeptides. In other embodiments, the nucleic acid encodes a SIP-specific fusion polypeptide comprising (i) two or more components which comprise a SIP binding domain of an immunoglobulin, and (ii) a multimerizing component.

In a related sixth aspect, the invention features a vector comprising a nucleic acid sequence of the invention. The invention further features an expression vector comprising a nucleic acid of the invention, wherein the nucleic acid molecule is operably linked to an expression control sequence. Also provided is a host-vector system for the production of the fusion polypeptides and trapbodies of the invention which comprises the expression vector of the invention which has been introduced into a host cell or organism, including, but not limited to, transgenic animals, suitable for expression of the fusion polypeptides and trapbodies. Suitable host cells include, for example, bacterial cells, e.g., E. coli, yeast cells, e.g., Pichia pastoris, insect cells, e.g., Spodoptera frugiperda, or mammalian cells, such as CHO or COS.

In a related seventh aspect, the invention features a method of producing a fusion polypeptide or trapbody of the invention, comprising culturing a host cell transfected with a vector comprising a nucleic acid sequence of the invention, under conditions suitable for expression of the protein from the host cell, and recovering the fusion protein or trapbody so produced.

In an eighth aspect, the invention features pharmaceutical compositions comprising a trapbody of the invention with a pharmaceutically acceptable carrier. Such pharmaceutical compositions may comprise trapbodies or nucleic acids which encode them.

In a ninth aspect, the invention features diagnostic and prognostic methods, as well as kits, for detecting, quantifying, and/or monitoring target molecule levels using a trapbody of the invention.

Other objects and advantages will become apparent from a review of the ensuing detailed description.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus for example, references to “a method” include one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference.

General Description

The invention encompasses fusion polypeptides, and, in specific embodiments, trapbodies, capable of binding and inhibiting the activity of soluble, interacting proteins (“SIPs”). The trapbodies are multimers, preferably dimers, of SIP-specific fusion polypeptides which comprise SIP binding domains derived from SIP targets and/or anti-SIP immunoglobulins, as well as multimerizing components. Generally, the multimerizing component is any component capable of multimerizing, e.g., forming a higher order complex of two or more fusion proteins. In the simplest embodiment, the SIP-specific fusion polypeptides which form the trapbody are comprised of TBD-IBD-M or IBD-IBD-M, where the SIP-specific components of each fusion polypeptide are directed to the same SIP. In a preferred embodiment of IBD-IBD-M, each immunoglobulin domain is specific to a different epitope of the same SIP (IBD′-IBD″-M).

Definitions

By the term “soluble interacting protein (SIP)” is meant any protein that is secreted or is circulating and which binds a receptor target and acts as an agonist or an antagonist. SIPs include, but are not limited to, cytokines, hormones, and growth factors.

By the term “soluble interacting protein target (SIP target)” is meant any protein which, when bound by a SIP, is either agonized (activated) or antagonized (inactivated).

By the term “SIP binding domain of a SIP target or TBD” is meant the entire SIP target, including, but not limited to, SIP binding proteins or full length receptors, as well as soluble (extracellular) domains thereof, as well as portions or functionally equivalent derivatives of such targets which bind to a SIP. This is intended to include not only the complete wild-type domain, but also insertional, deletional, and substitutional variants thereof, which substantially retain the functional characteristics of the intact domain. It will be readily apparent to one of skill in the art that numerous variants of the above SIP target-binding domain can be obtained which will retain substantially the same functional characteristics as the wild-type domain. Preferred amino acid substitutions, considered “conservative” or functionally similar, are described in WO 03013577A2 and Ausubel et al., eds., Current Protocols in Molecular Biology, Vol. 1, 1987 pg A.1C.1-A.1C.11

By the term “SIP binding domain of an immunoglobulin (immunoglobulin binding domain or IBD)” is meant that portion of an immunoglobulin that binds to a SIP, or functionally equivalent derivatives which bind a SIP. Thus, the term immunoglobulin binding domain, as used herein, includes full antibodies, as well as antibody fragments either produced by the modification of whole antibodies (e.g. enzymatic digestion), or those synthesized de novo using recombinant DNA methodologies [e.g., single chain Fv (scFv) (U.S. Pat. No. 5,969,108), single variable domains (Dabs) (Nuttall et al (2000) Current Pharm. Biotech. 1, 253-263) or those identified using display libraries, such as phage, E. coli or yeast display libraries (see, for example, McCafferty et al. (1990) Nature 348:552-554). Immunoglobulin binding domains also include, but are not limited to, the variable regions of the heavy (V_(H)) or the light (V_(L)) chains of immunoglobulins. Methods for producing such variable regions are described in Reiter et al. (1999) J. Mol. Biol. 290:685-698. IBD molecules include configurations such as scfv (U.S. Pat. No. 4,946,778), single-chain diabody molecules (Todorovska et al. (2001) J 1 mm. Methods 248;47-66 and Alt et al, (1999) FEBS Lett. 454:90-94) and stabilized scfv molecules (Auf der Maur et al (2001) FEBS Lett. 508:407-12) and single variable domain molecules (WO04041865A2). This is intended to include not only the wild-type domain, also insertional, deletional, and substitutional variants thereof which substantially retain the functional characteristics of the intact domain. Preferred amino acid substitutions, considered “conservative” or functionally similar, are described in WO 03013577A2 and Ausubel et al. supra. It will be readily apparent to one of skill in the art that numerous variants of the immunoglobulin binding domain can be obtained which will retain substantially the same functional characteristics as the wild-type domain.

Fusion Polypeptide Components

In specific embodiments, the SIP-specific polypeptides of the invention comprise a multimerizing component (M), which includes any natural or synthetic sequence capable of interacting with another multimerizing component to form a higher order structure, e.g., a dimer, a trimer, etc. In one embodiment, the multimerizing component comprises one or more immunoglobulin-derived domain from human IgG, IgM or IgA, or comparable immunoglobulin domains from other animals including, but not limited to, mice. In specific embodiments, the immunoglobulin-derived domain is selected from the group consisting of the Fc domain of IgG, the heavy chain of IgG, and the light chain of IgG. The Fc domain of IgG may be selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group. In a preferred embodiment, M is the Fc domain of IgG1, or a derivative thereof which may be modified for specifically desired properties. In addition, the multimerizing component may be unrelated to immunoglobulins and be, for example, a leucine zipper, a helix loop motif, or a coiled-coil motif.

Spacers

The term “spacer” or “linker” means one or more molecules, e.g., nucleic acids or amino acids, or nonpeptide moieties, such as polyethylene glycol, which may be inserted between one or more component domains. For example, spacer sequences may be used to provide a restriction site between components for ease of manipulation. A spacer may also be provided to enhance expression of the fusion protein from a host cell, to decrease steric hindrance such that the component may assume its optimal tertiary or quaternary structure and/or interact appropriately with its target molecule. For spacers and methods of identifying desirable spacers, see, for example, George et al. (2003) Protein Engineering 15:871-879, herein specifically incorporated by reference. One example of a spacer is (G₄S)₃ (US5158498) (SEQ ID NO:2) encoded by the nucleotide sequence of SEQ ID NO:1.

SIP Binding Immunoglobulins and Binding Domains

The SIP-specific fusion polypeptides and trapbodies of the invention comprise one or more immunoglobulin binding domains isolated from antibodies generated against a selected SIP. The term “immunoglobulin or antibody” as used herein refers to a mammalian, including human, polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen, which, in the case of the present invention, is a SIP or portion thereof. If the intended trapbody will be used as a mammalian therapeutic, immunoglobulin binding regions should be derived from the corresponding mammalian immunoglobulins. If the trapbody is intended for non-therapeutic use, such as for diagnostics and ELISAs, the immunoglobulin binding regions may be derived from either human or non-human mammals, such as mice. The human immunoglobulin genes or gene fragments include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regions, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Within each IgG class, there are different isotypes (eg. IgG₁, IgG₂, etc.) as well as allotypes thereof. Typically, the antigen-binding region of an antibody will be the most critical in determining specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit of human IgG, comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light chain (about 25 kD) and one heavy chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms “variable light chain” (V_(L)) and variable heavy chain (V_(H)) refer to these light and heavy chains respectively.

Antibodies exist as intact immunoglobulins, or as a number of well-characterized fragments produced by digestion with various peptidases. For example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′₂, a dimer of Fab which itself is a light chain joined to V_(H)-C_(H) by a disulfide bond. The F(ab)′₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the terms antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv (scFv) single variable domains (Dabs)) or those identified using display libraries such as phage, E. coli or yeast display libraries (see, for example, McCafferty et al. (1990) Nature 348:552-554). In addition, the SIP binding component of the fusion polypeptides and trapbodies of the invention include the variable regions of the heavy (V_(H)) or the light (V_(L)) chains of immunoglobulins, as well as SIP binding portions thereof. Methods for producing such variable regions are described in Reiter et al. (1999) J. Mol. Biol. 290:685-698.

Methods for preparing antibodies are known to the art. See, for example, Kohler & Milstein (1975) Nature 256:495-497; Harlow & Lane (1988) Antibodies: a Laboratory Manual. Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.). Antibodies that are isolated from organisms other than humans, such as mice, rats, rabbits, cows, can be made more human-like through chimerization or humanization.

“Humanized” or chimeric forms of non-human (e.g., murine) antibodies are immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′) 2 or other antigen-binding subsequences of antibodies) that contain minimal sequences required for antigen binding derived from non-human immunoglobulin. They have the same or similar binding specificity and affinity as a mouse or other nonhuman antibody that provides the starting material for construction of a chimeric or humanized antibody. Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin gene segments belonging to different species. For example, the variable (V) segments of the genes from a mouse monoclonal antibody may be joined to human constant (C) segments, such as IgG1 and IgG4. Human isotype IgG1 is preferred. A typical chimeric antibody is thus a hybrid protein consisting of the V or antigen-binding domain from a mouse antibody and the C or effector domain from a human antibody. Humanized antibodies have variable region framework residues substantially from a human antibody (termed an acceptor antibody) and complementarity determining regions (CDR regions) substantially from a mouse antibody, (referred to as the donor immunoglobulin). See, Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861, U.S. Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539. In one embodiment, the invention features CDR regions from hIL-6-specific antibodies selected from the group consisting of SEQ ID NOs:19-24, 27-32, 35-40, 43-48, 51-56, 59-64, 67-72, 75-80, 83-88, 91-96, 99-104, 107-112. The constant region(s), if present, are also substantially or entirely from a human immunoglobulin. The human variable domains are usually chosen from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable region domains from which the CDRs were derived. The heavy and light chain variable region framework residues can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See WO 92/22653. Certain amino acids from the human variable region framework residues are selected for substitution based on their possible influence on CDR conformation and/or binding to antigen. Investigation of such possible influences is by modeling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids. For example, when an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid should usually be substituted by the equivalent framework amino acid from the mouse antibody when it is reasonably expected that the amino acid: (1) noncovalently binds antigen directly; (2) is adjacent to a CDR region; (3) otherwise interacts with a CDR region (e.g. is within about 6 A of a CDR region), or (4) participates in the V_(L)-V_(H) interface. Other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. These amino acids can be substituted with amino acids from the equivalent position of the mouse donor antibody or from the equivalent positions of more typical human immunoglobulins. Other candidates for substitution are acceptor human framework amino acids that are unusual for a human immunoglobulin at that position. The variable region frameworks of humanized immunoglobulins usually show at least 85% sequence identity to a human variable region framework sequence or consensus of such sequences.

Fully human antibodies may be made by any method known to the art. For example, U.S. Pat. No. 6,596,541 describes a method of generating fully human antibodies. Briefly, initially a transgenic animal such as a mouse is generated that produces hybrid antibodies containing human variable regions (VDJ/VJ) and mouse constant regions. This is accomplished by a direct, in situ replacement of the mouse variable region (VDJ/VJ) genes with their human counterparts. The mouse is then exposed to human antigen, or an immunogenic fragment thereof. The resultant hybrid immunoglobulin loci will undergo the natural process of rearrangements during B-cell development to produce hybrid antibodies having the desired specificity. The antibody of the invention is selected as described above. Subsequently, fully-human antibodies are made by replacing the mouse constant regions with the desired human counterparts. Fully human antibodies can also be isolated from mice or other transgenic animals such as cows that express human transgenes or minichromosomes for the heavy and light chain loci. (Green (1999) J Immunol Methods. 231:11-23 and Ishida et al (2002) Cloning Stem Cells. 4:91-102) Fully human antibodies can also be isolated from humans to whom the protein has been administered. Fully human antibodies can also be isolated from immune compromised mice whose immune systems have been regenerated by engraftment with human stem cells, splenocytes, or peripheral blood cells (Chamat et al (1999) J Infect Dis. 180:268-77). To enhance the immune response to the protein of interest one can knockout the gene encoding the protein of interest in the human-antibody-transgenic animal.

The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, either directly from the producing B cells from the blood, lymph node, spleen, etc or from hybridomas made from the B cells or from EBV immortalized B cells using standard technologies. Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Pat. Nos. 4,946,778 and 4,816,567) can be adapted to produce antibodies used in the fusion proteins and methods of the instant invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express human or humanized antibodies.

Alternatively, phage display or related display technologies can be used to identify antibodies, antibody fragments, such as variable domains, and heteromeric Fab fragments that specifically bind to selected antigens. Gene libraries encoding heavy and light chains of monoclonal antibodies can be made from the hybridoma, spleen, lymph node or plasma cells described above or from naive, vaccinated, or diseased human sources of B cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity. Phage display is of particular value to isolate weakly binding antibodies or fragments thereof from unimmunized animals which, when combined with other weak binders in accordance with the invention described herein, create strongly binding trapbodies.

Screening and selection of preferred immunoglobulins (antibodies) can be conducted by a variety of methods known to the art. Initial screening for the presence of monoclonal antibodies specific to a SIP may be conducted through the use of ELISA-based methods or phage display, for example. A secondary screen is preferably conducted to identify and select a desired monoclonal antibody for use in construction of the trapbodies of the invention. Secondary screening may be conducted with any suitable method known to the art. One preferred method, termed “Biosensor Modification-Assisted Profiling” (“BiaMAP”) is described in U.S. Patent application 2004/101920, herein specifically incorporated by reference in its entirety. BiaMAP allows rapid identification of hybridoma clones producing monoclonal antibodies with desired characteristics. More specifically, monoclonal antibodies are sorted into distinct epitope-related groups based on evaluation of antibody: antigen interactions. Alternatively, ELISA-based, bead-based, or Biacore-based competition assays can be used to identify SIP binding pairs that bind different epitopes on the SIP and thus are likely to cooperate to bind the ligand with high affinity.

Nucleic Acid Construction and Expression

Individual components, SIP-specific fusion proteins, and the trapbodies of the invention may be produced from nucleic acids molecules using molecular biological methods known to the art. When the nucleic acids encode fusion polypeptides which comprise (i) one or more components which comprise a SIP binding domain of a SIP target; (ii) one or more components which comprise a SIP binding domain of an immunoglobulin and (iii) a multimerizing component, the multimerizing component multimerizes with a multimerizing component on another fusion polypeptide to form a trapbody or mini-trapbody of the invention. In other embodiments, the nucleic acid encodes fusion polypeptides comprising (i) two or more components which comprise a SIP binding domain of an immunoglobulin, and (ii) a multimerizing component, and the encoded polypeptides multimerize to form the trapbodies of the invention. Nucleic acid molecules are inserted into a vector that is able to express the fusion proteins when introduced into an appropriate host cell. Appropriate host cells include, but are not limited to, bacterial, yeast, insect, and mammalian cells. Any of the methods known to one skilled in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors encoding the fusion proteins of the invention under control of transcriptional/translational control signals. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinations (See Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, N.Y.).

Expression of the nucleic acid molecules of the invention may be regulated by a second nucleic acid sequence so that the molecule is expressed in a host transformed with the recombinant DNA molecule. For example, expression of the nucleic acid molecules of the invention may be controlled by any promoter/enhancer element known in the art. Promoters which may be used to control expression of the fusion polypeptide molecules include, but are not limited to, the long terminal repeat as described in Squinto et al. (1991) Cell 65:1-20; the SV40 early promoter region, the CMV promoter, the M-MuLV 5′ terminal repeat the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus, the herpes thymidine kinase promoter, the regulatory sequences of the metallothionine gene; prokaryotic expression vectors such as the b-lactamase promoter, or the tac promoter (see also “Useful proteins from recombinant bacteria” in Scientific American (1980) 242:74-94); promoter elements from yeast or fungi such as the Gal 4 promoter, the ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and tissue-specific transcriptional control regions derived from elastase I gene, insulin gene, immunoglobulin gene, mouse mammary tumor virus, albumin gene, α-fetoprotein gene, α1-antitrypsin gene, β-globin gene, myelin basic protein gene, myosin light chain-2 gene, and gonadotropic releasing hormone gene.

V_(L) and V_(H) domains. In accordance with the invention, the nucleic acid constructs include regions which encode SIP binding domains of immunoglobulins. In general, such binding domains will be derived from heavy (V_(H)) or light (V_(L)) chain variable regions to form an ScFv molecule or single V_(L) or V_(H) domains, to form single variable domain binders. After identification and selection of antibodies, ScFvs, Dabs, or Fabs exhibiting desired binding characteristics, the variable regions of the heavy chains and/or light chains of each antibody is isolated, amplified, cloned and sequenced. Modifications may be made to the V_(H) and V_(L) nucleotide sequences, including additions of nucleotide sequences encoding amino acids and/or carrying restriction sites, deletions of nucleotide sequences encoding amino acids, or substitutions of nucleotide sequences encoding amino acids. Modifications may also be made in the construction of scFv sequences utilizing the isolated V_(H) and V_(L) domains. These sequences may be constructed in either V_(H)-V_(L) or V_(L)-V_(H) orientation. Furthermore, the preferred (G₄S)₃ linker sequence may be lengthened, shortened, or removed. In addition, a linker sequence encoding alternate amino acids may be introduced and modified as above. As an alternate construction, isolated variable domain sequences can be assembled into a single chain diabody format (Alt et al FEBS Letters (1999) 454: 90-94). In this construction the V_(H) domain of a first desired binding domain would be joined by sequences encoding a short (5 amino acids or less) linker to the V_(L) domain of a second desired binding domain (V_(H1)-V_(L2)). This would be accomplished via overlapping PCR in a manner similar to the construction of scFvs described above. A second VHNL cassette would then be constructed in an analogous manner consisting of the V_(H) domain of the second desired binding domain upstream of the V_(L) domain of the first desired binding domain (V_(H2)-V_(L1)). These two cassettes could then be subcloned by standard techniques into an expression vector that results in the first cassette 5′ to the second cassette and may be separated by an additional linker sequence. As an optional configuration of this single chain diabody, the order of the V_(H) and V_(L) domains may be reversed such that the first cassette is organized in a V_(L1)-V_(H2) fashion and the second cassette is organized V_(L2)-V_(H1).

SIP target-binding domains. In accordance with the invention, the nucleic acid constructs include components which encode SIP target-derived binding domains. In preferred embodiments, such targets are SIP receptors. After identification of SIP binding domains exhibiting desired binding characteristics, the nucleic acids that encode such domains are used in the nucleic acid constructs. Such nucleic acids may be modified, including additions of nucleotide sequences encoding amino acids and/or carrying restriction sites, deletions of nucleotide sequences encoding amino acids, or substitutions of nucleotide sequences encoding amino acids.

The nucleic acid constructs of the invention are inserted into an expression vector or viral vector by methods known to the art, wherein the nucleic acid molecule is operatively linked to an expression control sequence. Also provided is a host-vector system for the production of a trapbody of the invention, which comprises the expression vector of the invention, which has been introduced into a host cell suitable for expression of the trapbody. The suitable host cell may be a bacterial cell such as E. coli, a yeast cell, such as Pichia pastoris, an insect cell, such as Spodoptera frugiperda, or a mammalian cell, such as a COS, CHO, 293, BHK or NSO cell.

The invention further encompasses methods for producing the fusion polypeptides and trapbodies of the invention by growing cells transformed with an expression vector under conditions permitting production of the SIP-specific fusion polypeptides and recovery of the trapbodies so produced. Cells may also be transduced with a recombinant virus comprising the nucleic acid construct of the invention.

The fusion polypeptides and trapbodies may be purified by any technique, which allows for the subsequent formation of a stable SIP-specific protein. For example, and not by way of limitation, the fusion proteins may be recovered from cells either as soluble proteins or as inclusion bodies, from which they may be extracted quantitatively by 8M guanidinium hydrochloride and dialysis. In order to further purify the trapbodies, conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography or gel filtration may be used. The trapbodies may also be recovered from conditioned media following secretion from eukaryotic or prokaryotic cells.

Diagnostic, Screening and Detection Methods

The compositions of the instant invention may be used diagnostically as well as prognostically. For example, the SIP-specific fusion polypeptides of the invention may be used to detect the presence of a particular SIP in a biological sample in order to quantitate the SIP levels or to determine if a subject has elevated SIP levels. Further, a fusion polypeptide of the invention can be used to monitor levels of SIPs in a biological sample obtained from a subject. The SIP-specific fusion polypeptides of the invention can be used in methods known in the art relating to the localization and activity of SIPs.

The fusion polypeptides and trapbodies of the invention may also be used in in vitro or in vivo screening methods where it is desirable to detect and/or measure SIP levels. Screening methods are well known to the art and include cell-free, cell-based, and animal assays. In vitro assays can be either solid state or soluble. SIP detection may be achieved in a number of ways known to the art, including the use of a label or detectable group capable of identifying a SIP-specific fusion polypeptide, which has trapped or otherwise bound a SIP. Detectable labels are well developed in the field of immunoassays and may generally be used in conjunction with assays using the fusion polypeptides and trapbodies of the invention.

The SIP-specific fusion polypeptides of the invention may also be directly or indirectly coupled to a label or detectable group when desirable for the purpose it is being used. A wide variety of labels may be used, depending on the sensitivity required, ease of conjugation, stability requirements, available instrumentation, and disposal provisions.

Therapeutic Methods

The fusion polypeptides and trapbodies of the invention can be used to ameliorate any disease or condition resulting from the action of one or more SIP molecules (“SIP-related condition or disease.”). These generally encompass diseases or conditions of a mammalian host, particularly a human host, which are associated with, or caused by, a particular SIP molecule. Thus, treating a SIP-related condition or disease will encompass the treatment of a mammal, in particular, human, who has symptoms reflective of increased SIP levels, or who is expected to have such elevated SIP levels in response to a disease, condition or treatment regimen. Treating a SIP-related condition or disease encompasses the use of a SIP-encoding nucleic acid of the invention to ameliorate an undesirable symptom resulting from the SIP-related condition or disease. The fusion polypeptides of the invention trap and inactivate the SIP molecule such that the SIP is prevented from attaching to SIP targets naturally circulating or present on cells in the subject being treated. In some circumstances, the trapbody accelerates clearance of the SIP from the circulation. Accordingly, administration of the SIP-specific fusion poypeptides of the invention to a subject suffering from a disease or condition associated with the activity or presence of a SIP results in amelioration or inhibition of the disease or condition. Further, the fusion polypeptides of the invention can be used prophylactically, both systemically and locally, to prevent an undesirable symptom or disease or condition from occurring or developing in a subject at risk for the development of the undesirable symptom, disease or condition. As used herein, a SIP-related condition also includes a condition in which it is desirable to alter, either transiently, or long-term, levels of a particular SIP in order to steer an immune response. SIP-related conditions also include those in which SIPs are administered therapeutically, wherein the trapbody of the invention is utilized to control SIP levels. In the treatment of a particular disease or condition associated with increased levels of one or more SIPS, the SIP-specific fusion polypeptides of the invention may be administered alone or in combination to effectively lower the level of the SIP(s).

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions comprising a fusion protein such as a trapbody of the invention and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

Kits

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with at least one SIP-specific fusion polypeptide of the invention. The kits of the invention may be used in any applicable method, including, for example, diagnostically. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human administration, (b) directions for use, or both.

Specific Embodiments

Example 1 illustrates one embodiment of the trapbodies of the invention, wherein the SIP is the cytokine IL-18. The trapbody is composed of different arrangements of a binding domain of the IL-18 receptor and/or an anti-IL-18 immunoglobulin-derived binding region, each with a multimerizing component. More specifically, fusion proteins were constructed with an IL-18 receptor component alpha (IL-18Ra) (SEQ ID NO:3), an anti-IL-18 scFv (SEQ ID NO:5), and a multimerizing component (hIgG1 Fc) (SEQ ID NO:8). Affinity determinations (Table 1) demonstrate that the combination of the specificity determining component of a cytokine receptor and a specific anti-cytokine antibody results in a molecule that has increased affinity for the cytokine over each component separately. Thus, they provide support for the use of the cytokine-specific fusion proteins of the present invention to reduce cytokine levels in diseases or conditions in which such cytokine levels are elevated.

Example 2 illustrates one embodiment of the trapbodies of the invention composed of anti-IL-6 scFv components to the same or different epitopes on the cytokine, each connected to a multimerizing component (Fc). As shown in Table 2, the bispecific scFv-scFv-Fc fusion proteins tested exhibited increased affinity (or affinity shift) for hIL-6 over that of the monospecific fusion proteins. The range of increased affinity is from ˜6 fold to 200-fold.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1 IL-18 Traps: Receptor-ScFv Fusion Proteins

Construction of Receptor-ScFv-Fc fusions. DNA constructs encoding the following orientations of cytokine receptor-scFv-Fc fusions were made: (1) the extracellular domain of human IL-18 receptor alpha (hIL-18Ra) (SEQ ID NO:4)-(G4S)₃ (SEQ ID NO:2)-anti-human IL-18 single chain Fv (anti-hIL-18 scFv) (SEQ ID NO:6)-Fc domain of human IgG1 (Fc) (SEQ ID NO:8); (2) anti-hIL-18 scFv (SEQ ID NO:6)-(G₄S)₃ (SEQ ID NO:2)-hIL-18Ra (SEQ ID NO:4)-Fc (SEQ ID NO:8); (3) hIL-18Ra (SEQ ID NO:4)-Fc (SEQ ID NO:8)-anti-hIL-18 scFv (SEQ ID NO:6); (4) anti-hIL-18 scFv (SEQ ID NO:6)-Fc (SEQ ID NO:8)-hIL-18Ra (SEQ ID NO:4); (5) hIL-18Ra (SEQ ID NO:4)-Fc (SEQ ID NO:8) and (6) anti-hIL-18 scFv (SEQ ID NO:6)-Fc.

The extracellular domain, nucleotides 76-1011 (amino acids 18-329) (SEQ ID NO:3) of human IL-18 receptor a (Genbank #U43672) was PCR cloned from spleen cDNA. The anti-human IL-18 ScFv was constructed by overlapping oligonucleotide synthesis and PCR that resulted in fusing the heavy chain variable region (nucleotides 58 to 396; amino acids 20-132, Genbank #AB017433) to a nucleotide sequence (SEQ ID NO:1) encoding the amino acid sequence, (G₄S)₃ (SEQ ID NO:2), fused to the light chain variable region (nucleotides 61-384 (amino acids 21-128; Genbank Accession #AB017434) (V_(H)-V_(L)) (SEQ ID NO:5). The Fc domain of human IgG1 comprises nucleotides 79-765 (amino acids 27-255) of the human IgG1 Fc domain (Genbank #X70421), containing a nucleotide change at nucleotide 82 (T to G) to change the amino acid at 28 from Cys to Gly (SEQ ID NO:7).

Mammalian Expression of Receptor-ScFv-Fc fusion proteins. DNA constructs encoding the above cytokine receptor-ScFv-Fc fusion proteins were transiently transfected into CHO cells by lipofectamine/LIPO plus (Life Technologies) and supernatants were collected after 72 hours. Protein expression was measured by Western blotting with anti-human Fc HRP-conjugated antibody (Promega) and visualized by ECL (Pierce).

Affinity Measurements by BIAcore. The affinity of the various cytokine receptor-scFv fusion proteins for human IL-18 was measured using a BIAcore 2000. Receptor-scFv fusion proteins present in the CHO supernatant were captured onto the chip surface using anti-human Fc antibodies. Various concentrations of human IL-18 were injected over the surface and the time course of association and dissociation was monitored. Kinetic analysis using BIA evaluation software was performed to obtain the association and dissociation rate constants.

Results. The results are shown in Table 1 below. All of the receptor-scFv-Fc fusions exhibited increased affinity over that of each component separately. The fusions with the Fc domain at the C-terminus exhibited an approximately 4-fold higher affinity for hIL-18 than the anti-hIL-18 ScFv alone and an approximately 75-fold higher affinity for hIL-18 than the hIL-18Ra alone. The fusions with the Fc domain in between the hIL-18Ra and the anti-hIL-18 scFv exhibited a 2-fold and 75-fold increase in affinity over the anti-hIL-18 scFv alone or the hIL-18Rα alone, respectively. TABLE 1 KD (pM) off rate s⁻¹ on rate M⁻¹ s⁻¹ anti-hIL18 ScFv-Fc 193 4.65 × 10⁻⁵ 2.41 × 10⁵ hIL18Rα-Fc 4180 6.53 × 10⁻³ 1.56 × 10⁶ hIL18Ra-anti-hIL18 ScFv-Fc 53 2.12 × 10⁻⁵   4 × 10⁵ anti-hIL18 ScFv-hIL18Rα-Fc 42 1.61 × 10⁻⁵ 3.87 × 10⁵ hIL18Rα-Fc-anti-hIL18 ScFv 92 3.70 × 10⁻⁵ 4.04 × 10⁵ anti-hIL18 ScFv-Fc-hIL18Rα 137 3.90 × 10⁻⁵ 1

Example 2 IL-6 Traps: ScFv-ScFv Constructs

Generation of anti-IL-6 antibodies. Mice (Balb/c) were immunized with human IL-6 (hIL-6) protein (Regeneron Pharmaceuticals, Inc., New York) in adjuvant once every three weeks for a total of three injections followed by a boost with hIL-6 one month later. Approximately 2 weeks later, the spleen cells from one mouse were fused with myeloma cells by electrofusion. After fusion, the hybridoma cells were grown as a pool, and then plated as single cells in 96-well plates. A primary screen for the presence of monoclonal antibodies specific for IL-6 was performed on the hybridoma cell supernatant by two different ELISA methods. In one format, the hIL-6 was coated directly onto microtiter plates, incubated with the hybridoma cell supernatant and the presence of positive antibodies was visualized with anti-mouse IgG-HRP conjugated antibodies. The second format utilized a biotinylated hIL-6 that was captured onto the surface of microtiter plates coated with neutravidin. The hybridoma cell supernatant was incubated in the wells and the presence of positive antibodies was visualized with anti-mouse IgG-HRP conjugated antibodies. Hybridoma cell supernatant was considered positive i.e., contained anti-hIL-6 monoclonal antibodies, if the OD at 450 nm was equal or greater than 1.0 in either ELISA format.

Binding assays. Cell supernatant for hybridomas expressing monoclonal antibodies that scored positive on the primary screen were then assayed in secondary screens for total antibody concentration and apparent affinity by a binding assay. For the binding assay, hybridoma cell supernatant containing antibodies at a concentration of 1 nM were incubated for 3 hours at room temperature with varying concentrations of hIL-6. These mixtures were assayed for the presence of free antibody using the hIL-6 ELISA from the primary screen. An apparent KD can be calculated from the IC₅₀, the IL-6 concentration at which half of the antibody is free. Based on this analysis, five hybridomas were chosen for cell cloning by limiting dilution.

Epitope analysis. Supernatant from 25 positive hybridomas from the primary and secondary screens were further analyzed by Bia-MAP and antibody competition assays to identify classes of antibodies that recognize different epitopes on hIL-6. The monoclonal antibodies grouped into 4 classes, with one class subdivided into 5 groups. Based on these data, 5 additional hybridoma cells representing antibodies from the different classes were chosen for ‘limiting dilution’ cloning.

Isolation of antibody variable regions. The variable region of the heavy chain (V_(H)) and the light chain (V_(L)) for each antibody was isolated from clonal hybridoma cells using rapid amplification of cDNA ends (RACE). Total RNA was isolated from ˜2-5×10⁷ hybridoma cells using Trizol reagent. The isotype for each of the monoclonal antibodies was determined and isotype-specific primers within the constant region of the V_(H) and V_(L) were utilized to synthesize the 1^(st) strand cDNA from total RNA. This cDNA was then tailed with polyG using terminal transferase and the variable regions were then amplified by PCR using a polyC primer and a second set of isotype-specific primers nested to the original set. The amplification product was then cloned and sequenced. The V_(H) and V_(L) for each antibody was constructed into a scFv format by PCR using standard techniques to insert a stretch of nucleotides between the 3′ end of the V_(H) and the 5′ end of the V_(L) that would encode for 15 amino acids, (G₄S)₃ (SEQ ID NO:2) as well as add nucleotides carrying restriction sites at the 5′ end of the V_(H) and 3′ end of the V_(L) for ease of cloning. Additionally, by use of an alternate set of 3′ V_(H) and 5′ V_(L) PCR primers a subset of scFvs were generated which lacked the (G₄S)₃ (SEQ ID NO:2) linker domain between the two variable domains. In these specific scFv's the 3′ end of the V_(H) was fused directly to the 5′ end of the V_(L). However, these scFv's may be optionally modified to include a smaller or larger linker sequence.

Construction of ScFv-ScFv-Fc fusions. All combinations of scFvs (having mono- or multispecificity) were constructed in a mammalian expression vector by restriction enzyme subcloning to generate two scFvs in tandem separated by nucleotide sequence SEQ ID NO:1 between the two scFvs followed by the coding region of Fc domain of human IgG1. A subset of additional scFv-scFv-Fc fusions were created in which the (G₄S)₃ linker removed scFv's were used in place of the standard scFv's. Furthermore, through the use of standard molecular techniques a final subset of scFv-scFv-Fc fusions were generated in which the (G₄S)₃ linker sequence between the individual scFv's was removed. Briefly, PCR amplification followed by sequence verification was utilized to replace the 3′ restriction site on the N-terminal scFv coding sequence with that used on the 5′ end of the C-terminal scFv sequence. Direct restriction subcloning of this fragment into an scFv-scFv-Fc expression vector resulted in (G₄S)₃ removal. This process was performed with scFv-scFv-Fc fusions which both retained and lacked the (G₄S)₃ sequence internal to each scFv. DNA constructs encoding various scFv-scFv-Fc fusion proteins were transiently transfected into CHO cells by lipofectamine (Life Technologies) by standard techniques and supernatants were collected after 72 hours. Protein expression was measured by Western blotting with anti-human Fc HRP-conjgated antibody (Promega) and visualized by ECL (Pierce).

Affinity Measurements. The affinity of the various scFv-scFv-Fc fusion proteins for human IL-6 was measured using a BIAcore 2000. ScFv-scFv-Fc fusion proteins present in the CHO cell supernatant from transient transfection were captured onto the chip surface that had been coated with anti-human Fc antibodies. Between 300-700 Resonance Units (RUs) were captured. After capture, various concentrations of hIL-6 (1.25 nM, 2.5 nM and 5 nM) were injected over the surface and the time course of association and dissociation was monitored. Kinetic analysis using BlAevaluation software was performed to obtain the association and dissociation rate constants. The results are shown in Table 2. TABLE 2 Fold Increase Fold Increase (from position (from position Construct ScFv Kd 1) 2) ScFv-Fc 8F10(SEQ ID 430 pM NO: 9, 10) 3D6(SEQ ID 1.2 nM NO: 11, 12) 6E2(SEQ ID 608 pM NO: 13. 14) 5A11(SEQ ID 1.5 nM NO: 15, 16) ScFv- 8F10.8F10 296 pM ScFv-Fc 3D6.3D6 3.3 nM 6E2.6E2 651 pM 3D6.3D6 3.1 nM 6E2.3D6 85 pM 7.7 38.8 3D6.6E2 30 pM 110.0 21.7 8F10.6E2 5.1 pM 58.0 127.6 6E2.8F10 28.0 pM 23.3 10.6 5A11.6E2 81 pM 38.3 8.0 6E2.5A11 92 pM 7.1 33.7 5A11.8F10 49 pM 63.3 6.0 5A11.3D6 16 pM 193.8 206.3 

1. An isolated nucleic acid encoding a SIP-specific fusion polypeptide, wherein the fusion polypeptide comprises: (a) one or more components which comprise a SIP binding domain of a SIP target (TBD); (b) one or more components which comprise a SIP binding domain of an immunoglobulin (IBD); and (c) a multimerizing component (M) capable of multimerizing with a multimerizing component on another fusion polypeptide to form a multimer of the fusion polypeptides.
 2. The isolated nucleic acid of claim 1, wherein the multimerizing component comprises a constant region(s) of an immunoglobulin or derivative thereof.
 3. The isolated nucleic acid of claim 2, wherein the multimerizing component is selected from the group consisting of the Fc domain of IgG, the Fc domain of the heavy chain of IgG, the Fc domain of the light chain of IgG, and the heavy chain CH² and CH³ constant regions.
 4. The isolated nucleic acid of claim 1, wherein the IBD is selected from the group consisting of a heavy chain variable domain, a light chain variable domain and an ScFv.
 5. The isolated nucleic acid of claim 1, wherein the SIP-specific fusion polypeptide component arrangements are selected from the group consisting of (TBD)_(x)-(IBD)_(y)-M; (TBD)_(x)-M-(IBD)_(y); (IBD)_(x)-(TBD)_(y)-M; (IBD)_(y)-M-(TBD)_(x), M-(IBD)_(y)-(TBD)_(x), and M-(TBD)_(x)-(IBD)_(y), wherein TBD is a SIP target binding domain, IBD is an immunoglobulin-derived SIP-binding domain, M is a multimerizing component, and x≧1 and y≧1.
 6. A fusion polypeptide encoded by the nucleic acid of any of claims 1 to
 5. 7. A trapbody comprising two of the fusion polypeptides of claim
 6. 8. An isolated nucleic acid encoding a SIP-specific fusion polypeptide, wherein the fusion polypeptide comprises: (a) two or more components which comprise a SIP binding domain of an immunoglobulin (IBD); and (b) a multimerizing (M) component.
 9. The isolated nucleic acid of claim 8, wherein the fusion polypeptide comprises IBD′-IBD″-M, wherein IBD′ and IBD″ are directed to different epitopes of the same SIP.
 10. The isolated nucleic acid of claim 8, wherein the SIP is a cytokine.
 11. The isolated nucleic acid of claim 10, wherein the SIP target is a specificity-determining component of a cytokine receptor or a signaling component of a cytokine receptor.
 12. The isolated nucleic acid of claim 11, wherein the cytokine is selected from the group consisting of interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-13, IL-15, IL-18, granulocyte macrophage colony stimulating factor, oncostatin M, leukemia inhibitory factor, ciliary neurotrophic factor, MIF, interferon gamma, and cardiotrophin-1.
 13. A fusion polypeptide encoded by the nucleic acid molecule of any of claims 8 to
 12. 14. A trapbody which is a dimer comprising two of the fusion polypeptides of claim
 13. 15. A method of producing a SIP-specific fusion polypeptide, comprising culturing a host cell transfected with a vector comprising the nucleic acid of claim 1 or 8, under conditions suitable for expression of the protein from the host cell, and recovering the fusion protein so produced.
 16. An isolated nucleic acid encoding (i) a V_(L) CDR selected from the group consisting of SEQ ID NOs:27-32, 43-48, 59-64, 75-80, 91-96 and 107-112, and (i) a V_(H) CDR selected from the group consisting of SEQ ID NOs:19-24, 35-40, 51-56, 67-72, 83-88, and 99-104.
 17. An IL-6-specific antibody or antibody fragment, comprising at least one CDR of claim
 16. 18. An IL-6-specific fusion polypeptide comprising two IL-6-specific immunoglobulin domains and a multimerizing component, wherein each immunoglobulin domain comprises at least one CDR of claim
 16. 19. The IL-6-specific fusion polypeptide of claim 18, wherein at least one immunoglobulin domain is humanized.
 20. A trapbody comprising two IL-6-specific fusion polypeptides of claim 18 or claim
 19. 21. A trapbody, comprising two fusion polypeptides and capable of binding a soluble protein (SIP), wherein each fusion polypeptide comprises IBD′-IBD″-M, wherein IBD′ and IBD″ are specific to different epitopes of the same SIP, and the trapbody exhibits at least a five-fold increase in affinity relative to a dimer composed of two fusion polypeptides each having a single IBD and a multimerizing component.
 22. A human IL-6-specific trapbody, comprising IBD-IBD-M, wherein each IBD comprise at least one CDR sequence selected from the group consisting of SEQ ID NOs:19-24, 27-32, 35-40, 43-48, 51-56, 59-64, 67-72, 75-80, 83-88, 91-96, 99-104 and 107-112.
 23. The IL-6-specific trapbody of claim 22, wherein each IBD comprises (i) at least one V_(L) CDR selected from the group consisting of SEQ ID NOs:27-32, 43-48, 59-64, 75-80, 91-96 and 107-112, and (i) at least one V_(H) CDR selected from the group consisting of SEQ ID NOs:19-24, 35-40, 51-56, 67-72, 83-88, and 99-104.
 24. An antibody heavy chain or heavy chain fragment, comprising one or more of CDR1, CDR2 and CDR3, wherein (i) CDR1 is selected from the group consisting of SEQ ID NO:19, 22, 35, 38, 51, 54, 67, 70, 83, 86, 99 and 102; (ii) CDR2 is selected from the group consisting of SEQ ID NO: 20, 23, 36, 39, 52, 55, 68, 71, 84, 87, 100 and 103; and (iii) CDR3 is selected from the group consisting of SEQ ID NO: 21, 24, 37, 40, 53, 56, 69, 72, 85, 88, 101 and
 104. 25. The antibody heavy chain or heavy chain fragment of claim 24 which is humanized.
 26. An antibody light chain or light chain fragment, comprising one or more of CDR1, CDR2 and CDR3, wherein (i) CDR1 is selected from the group consisting of SEQ ID NO: 27, 30, 43, 46, 59, 62, 75, 78, 91, 94, 107 and 110; (ii) CDR2 is selected from the group consisting of SEQ ID NO: 28, 31, 44, 47, 60, 63, 76, 79, 92, 95, 108, and 111; and (iii) CDR3 is selected from the group consisting of SEQ ID NO: 29, 32, 45, 48, 61, 64, 77, 80, 93, 96, 109 and
 112. 27. The antibody light chain or light chain fragment of claim 26 which is humanized.
 28. An antibody or antibody fragment, comprising the heavy chain or heavy chain fragment of claim 24 and the light chain or light chain fragment of claim
 26. 29. The antibody or antibody fragment of claim 28 which is humanized. 